SCAS Sumika Chemical 

. Analysis Service 

Report 

Study Title 

1-7-5, Kikumoto-cho 

Niihama-shi, Ehime 792-0801 JAPAN 

Tel: +81-897-32-3411 

Fax: +81-897-32-9644 

Determination of physico-chemical properties of 

·Hydrolysis as a Function of pH 

·Partition Coefficient (n-octanollwater) 

Study Director 

Shuichiro Manabe 

Study Completion Date 

May 7, 2013 

Test Facility 

Sumika Chemical Analysis Service, Ltd. 

1-7-5 Kikumoto-cho, Niihama-shi, Ehime, 792-0801 Japan 

Study Number 

8316490-00 

Sponsor 

Sumika Chemical Analysis Service, Ltd. 
Study Number:8316490-00 

Signatures 

Operator Tsuyoshi Hamada 

Date: 

Study Director Shuichiro Manabe 

Date: 

1120 
Study Number:8316490-00 

Contents 

1. Purpose of the study ...................................................................................................................... 3 

2. Sample information ....................................................................................................................... 3 

3. Summary ........................................................................................................................................ 4 

4. Hydrolysis as a Function of pH ..................................................................................................... 5 

4.1. Guideline ................................................................................................................................. 5 

4.2. Principle of the test ................................................................................................................ 5 

4.3. Performance of the preliminary test ..................................................................................... 5 

4.3.1. Preparation of apparatus and reagents ......................................................................... 5 

4.3.1.1 Preparation of apparatus .......................................................................................... 5 

4.3.1.2 Preparation of buffer solutions ................................................................................. 5 

4.3.1.3 Preparation of mobile phase ..................................................................................... 6 

4.3.1.4 Preparation of standard solutions ........................................................................... 6 

4.3.2. Pretreatment ................................................................................................................... 6 

4.3.3. Analysis method .............................................................................................................. 6 

4.4. Data handling ......................................................................................................................... 6 

4.5. Results ..................................................................................................................................... 7 

5. Partition coefficient (n-octanol/water) .......................................................................................... 8 

5.1. Guideline ................................................................................................................................. 8 

5.2. Principle of the test ................................................................................................................ 8 

5.3. Preparation of solutions ......................................................................................................... 8 

5.3.1. Sample solutions .............................................................................................................. 8 

5.3.2. Standard solutions .......................................................................................................... 8 

5.3.3. Dead Time solution ......................................................................................................... 8 

5.4. Analysis method ..................................................................................................................... 9 

5.5. Data handling ......................................................................................................................... 9 

5.6. Results ................................................................................................................................... 10 

6. Tables ............................................................................................................................................ 11 

6.1. Hydrolysis as a Function of pH ........................................................................................... 11 

6.2. Partition coefficient (n-octanollwater) ................................................................................. 12 

7. Figures .......................................................................................................................................... 13 

7.1. Hydrolysis as a function of pH ............................................................................................. 13 

7.2. Partition coefficient (n-octanol/water) ................................................................................. 17 

2/20 
Study Number:8316490-00 

1. Purpose of the study 

The purpose of the study was to determine the following physico-chemical properties for 

·Hydrolysis as a Function of pH 

·Partition coefficient (n-octanollwater) 

2. Sample information 

Chemical Name: 

Structure Formula: 

Identification: 

CAS No.: 

Purity: 

Lot No.: 

Appearance: 

Storage: 

3/20 
Study Number:8316490-00 

3. Summary 

Property Guideline Result Method 

Hydrolysis rate 

(50 "C, 5 days) 

Hydrolysis as a 

OECD 111 -

Function of pH < 10% (pH=4) 

< 10% (pH=7) 

< 10% (pH=9) 

Partition 

coefficient OECD 117 > 5.7 HPLCmethod 

(n-octanol/water) 

4/20 
4. Hydrolysis as a Function of pH 

4.1. Guideline 

The study was based on the following guideline: 

Study Number:8316490-00 

Organization for Economic Co-operation and Development (OECD), OECD Guidelines for the 

Testing of Chemicals no. 111: "Hydrolysis as a Function of pH", April13, 2004. 

4.2. Principle of the test 

Sterile aqueous buffer solutions of different pH values (pH 4, 7 and 9) are treated with the 

sample and incubated in the dark under controlled laboratory conditions (at constant 

temperatures). 

After appropriate time intervals, buffer solutions are analyzed for the sample and for 

hydrolysis products. 

4.3. Performance of the preliminary test 

4.3.1. Preparation of apparatus and reagents 

4.3.1.1 Preparation of apparatus 

The glassware to be used was heated by drying machine. (170 - 180 'C, 1 hour) 

The volumetric flasks which was after washed with 70 % ethanol and naturally dried 

were used. 

4.3.1.2 Preparation of buffer solutions 

(1) pH 4 buffer solution (0.1mol/ L phthalic acid buffer solution) 

400 mL of water that was passing nitrogen for 5 min, 10.2 g of potassium hydrogen 

phthalate and 4 mL of 0.1mo1/ L sodium hydroxide were taken to the flask. Then water 

was added to prepare to 500mL, and stirred. 

(2) pH 7 buffer solution (0.1mol/ L phosphate buffer solution) 

400 mL of water that was passing nitrogen for 5 min, 6.8 g of potassium dihydrogen 

phosphate and 29.6 mL of 1mol I L sodium hydroxide were taken to the flask. Then 

water was added to prepare to 500mL, and stirred. 

(3) pH 9 buffer solution (0.1mol/ L borate buffer solution) 

400 mL of water that was passing nitrogen for 5 min, 3.1 g of boric acid and 21.3 mL of 

1mo1/ L sodium hydroxide were taken to the flask. Then water was added to prepare to 

500mL, and stirred. 

Buffer solutions above were heated in an autoclave. ( 121 'C, 20 minutes) 

5/20 
Study Number:8316490-00 

Preparation of mobile phase 

The mobile phase was prepared to pH 2.1 by adding 0.1mol I L phosphoric acid solution 

to a solution of sodium dihydrogen 0.1mol I L phosphate. 

4.3.1.4 Preparation of standard solutions 

The standard solutions were prepared by diluting with the mobile phase within the 

range of 0.14 to 3.6 giL. 

4.3.2. Pretreatment 

The sample were weighed and placed into each of 6 volumetric flasks (50 mL) and 

appropriate amount of 1mo1 I L sodium hydroxide were added to each flasks. Then they were 

filled up to the marked line with buffer solutions. Two flasks each (pH 4, 7 and 9) were 

prepared at a concentration of 30 giL (approximately 0.01 mol I L) with buffer solutions. 

The solutions were stood for 5 days in a water bath (50 ± 0.5 "C) and filtered through a 

0.2-micrometer membrane filter, then the filtrates were diluted 20 times with mobile phase. 

The solutions were used as measurement solution. 

4.3.3. Analysis method 

The solutions were analyzed with the GPC method. 

GPC conditions are shown below. 

<GPC conditions> 

Apparatus: 

Separation columns: 

LC-10ATVP (Shimadzu) 

Eluent: 

Flow rate: 

Detector: 

Column temperature: 

Injection volume: 

4.4. Data handling 

Sample concentration of the exposed solution, hydrolysis rates of the sample and spike 

recoveries of the sample were calculated by the following equations: 

6/20 
Y=aX+b 

C=f· X 

G = (C-D)iD • 100 

H = (E-D)/D · 100 

Y = Peak height (m V) 

X= Concentration of measurement solution (giL) 

a= Slope 

b = y-Axis intercept 

f =Dilution factor 

C = Sample concentration of the exposed solution for 0 days. 

: Calculated from the measured values (giL) 

D = Sample concentration of the exposed solution 

Study Number:8316490-00 

: Calculated from the amount of the adjusted the sample (giL) 

E =Sample concentration of the exposed solution for 5 days. 

: Calculated from the measured values (giL) 

G = Spike recoveries (%) 

H =Hydrolysis rate (%) 

4.5. Results 

The results of standard solution measurement, spike recoveries and hydrolysis rate are 

shown in Table 1, 2 and 3. 

The calibration curve is shown in Figure 1. 

The typical GPC chromatograms are shown in Figure 2 to 12 respectively. In each pH 

condition, spike recoveries were the average value of 98% to 102% and variations were 0.26% 

to 6.8%. 

In conclusion, the hydrolysis rate ofthe sample was determined to be <10 %. Therefore, there 

is not necessary of the further hydrolysis examination. 

7/20 
5. Partition coefficient (n-octanolfwater) 

5.1. Guideline 

The study was based on the following guideline: 

Study Number:8316490-00 

Organization for Economic Co-operation and Development (OECD), OECD Guidelines for the 

Testing of Chemicals no. 117: "Partition Coefficient (n-octanolfwater)", Aprill3, 2004. 

5.2. Principle of the test 

Reverse phase HPLC IS performed on analytical columns packed with a commercially 

available solid phase containing long hydrocarbon chains chemically bound onto silica. 

Hydrophilic chemicals are eluted first and lipophilic chemicals are eluted last. Elution 

corresponds to a partition coefficient (n-octanolfwater). 

Partition coefficient (n-octanolfwater) of the sample is calculated with the retention time of 

standard materials. 

5.3. Preparation of solutions 

5.3.1. Sample solutions 

The sample was dissolved in mixture of borate buffer (pH9.8) I methanol (25/75) to obtain a 

sample solution at a concentration of 500~ 1000 mg/L. Then the solution was filtered through 

a 0.45-micrometer membrane filter. 

As a blank solution, mobile phase was used. 

5.3.2. Standard solutions 

Aniline, toluene, naphthalene, ethyl benzene, biphenyl and triphenylamine were chosen as 

reference items from the list given in the OECD test guideline no.l17. They were dissolved 

into methanol to prepare a standard stock solution at a concentration of 520 to 760 mg/L. The 

standard stock solutions were mixed and diluted with borate buffer (pH9.8) I methanol 

(25/75) to obtain a standard solution at a concentration between 26 and 38 mgiL respectively. 

5.3.3. Dead Time solution 

The stock solution of thiourea was prepared at a concentration of 590 mgiL with borate 

buffer (pH9.8) I methanol (25/75). The dead time solution was prepared by diluting the stock 

solution to a concentration of 29 mg/L with borate buffer (pH9.8) I methanol (25/75). 

8/20 
5.4. Analysis method 

Apparatus: 

Study Number:8316490-00 

LC-lOATVP (Shimadzu) 

Column: 

Mobile Phase: 

UV Detector: 

Injection Volume: 

Flow Rate: 

Column Temperature: 

5.5. Data handling 

The capacity factor is described by the following equation. 

k = (tR-to)lto 

where 

k = Capacity factor 

tR =Retention time of the sample and the standard materials 

to = Retention time of the dead time material 

The partition coefficient (n-octanollwater) of the sample is calculated by experimentally 

determining its capacity factor and then inputting k into the following equation. 

Y=a·X+b 

where 

Y =Partition coefficient (n-octanol/water) (logP) 

X= logk 

a= Slope 

b = y-Axis intercept 

9/20 
Study Number:8316490-00 

5.6. Results 

The calibration data of partition coefficient (n-octanol/water) are shown in Table 4. 

The calibration curve is shown in Figure 13. The coefficient of determination (r2) was 0.963. 

In the case with no column, the increase of peak area of the sample solution was detected 

compared with the blank solution. However in the case with a column, there was no significant 

difference in peak area between the sample solution and the blank solution. It presumed that 

the main components of was adsorbed in the reversed phase HPLC column and not 

eluted. Therefore, logP of the sample was presumed 5. 7 or more. 

The typical chromatograms of the dead time solution, the standard solution, the sample 

solution (with no column and with a column) and the blank solution (with no column and with 

a column) are shown in Figures 14, 15, 16 and 17 respectively. 

In conclusion, the partition coefficient (n-octanol/water), logP, of the sample was presumed 

5.7 or more. 

10/20 
Study Number:8316490-00 

6. Tables 

6.1. Hydrolysis as a Function of pH 

Table 1 The results of standard solution measurement 

No. Concentration (giL) Height (mV) 

1 0.14 13 

2 0.56 52 

3 1.4 132 

4 3.6 325 

Table 2 Boike recoveries 

Sample 

Sample 

concentration 

Preparation concentration Spike Difference 

Liquid Dilution Height in the Average Variation 

No. concentration in the liquid recoveries between 

exposure ratio (mV) measured (%) (%) 

(g/L) exposure (%) n1 and n2 

solution 

(g/L) 

(g/L) 

0 day n=l 30 21 135 1.5 30 101 

0.02 101 0.02 

pH 4 n=2 30 21 135 1.5 31 101 

0 day n=l 30 21 132 1.4 30 100 

4.1 98 4.1 

pH 7 n=2 30 21 126 1.4 29 96 

0 day n=l 30 21 130 1.4 30 100 

7.3 103 7.0 

pH 9 n=2 30 21 139 1.5 32 107 

Table 3 Hvdrolvsis rate 

Sample Sample 

Preparation concentration concentration Hydrolysis 

Liquid Dilution Height 

No. concentration in the measured in the liquid rate 

exposure ratio (mV) 

(g/L) solution exposure (%) 

(g/L) (g/L) 

5 days n=1 30 20 133 1.5 30 <10 

pH 4 n=2 30 21 139 1.5 31 (10 

5 days n;::;l 30 21 130 1.4 29 (10 

pH 7 n=2 30 21 136 1.5 31 <10 

5 days n=1 30 21 143 1.6 33 <10 

pH 9 n=2 30 21 139 1.5 32 <10 

ll/20 
Study Number:8316490-00 

6.2. Partition coefficient (n-octanol/water) 

Table 4 Calibration data of uartition coefficient (n -octanol/water) 

Dead time logk logPow Standard material to tR 

material <X> <Y> 

Thiourea 3.28 ~ ..___________ ~ 

Aniline ----- ~ 4.07 -0.616 0.9 

Toluene ----- ~ 8.81 0.228 2.7 

Naphthalene ----- ~ 10.68 0.354 3.6 

Ethylbenzene ~ ~ 11.30 0.389 3.2 

Biphenyl ---- ~ 14.64 0.540 4.0 

Triphenylamine ---- ~ 37.76 1.022 5.7 

12/20 
Study Number:8316490-00 

7. Figures 

7.1. Hydrolysis as a function of pH 

[mVl 

350 

300 

250 

fa> 200 

~ _§_ 150 

100 

50 .,. 0 

0.00 

y · 0:1:'7tXT1-:-730S 

R'-noooo ~ 

./" 

./" 

_/ 

../"" 

1.00 2.00 

Concentration 

(g/L) 

Figure 1 Calibration curve 

3.00 

_,. 

4.00 

300 00

. 

20[00

20. 00 

400. 000 

30. 00 

[min] 

Figure 2 GPC chromatogram of the standard solution (No.3) 

' 

' 

' 

' 

40. 00 

300. 000 -----------------------------------~------------------------------------

' 

' 

' 

' ' 200. 000 -----------------------------------~------------------------------------- ' 

20. 00 

' 

30. 00 

lminl 

Figure 3 GPC chromatogram ofthe mobile phase 

13/20 

40. 00 
Study Number:8316490-00 

[mV] 

[mVl 

[mV] 

40!000 -----------------------------------,------------------------------------ ' 

' 

' 

' 

' 300.000 ----------------------------------- '------------------------------------

20!000 ----------------------------------- ------------------------------------

20.00 

400. 000 

30. 00 

[min] 

Figure 4 GPC chromatogram of the buffer solution (pH 4) 

' 

\I 

40. 00 

300. 000 -----------------------------------,------------------------------------

' 

200. 000 -----------------------------------~ 

20. 00 

400. 000 

' y 

30. 00 

[mini 

Figure 5 GPC chromatogram of the buffer solution (pH 7) 

\I 

40. 00 

300.000 ----------------------------------- ------------------------------------

200. 000 -----------------------------------~------------------------------------

20. 00 

' 

' 

30. 00 

[mini 

Figure 6 GPC chromatogram of the buffer solution (pH 9) 

14/20 

\( 

40. 00 
Study Number:8316490-00 

[mVl 

[mVl 

[mV] 

400. 

300. 000 

20[000

1 00· ~8. 00 

lminl 

Figure 7 GPC chromatogram ofthe sample solution (0 day, pH 4 n=ll 

300.000 

20[000 

1 00. 008 

2.00 3[00 40. 00 

[min] 

Figure 8 GPC chromatogram of the sample solution (0 day, pH 7 n=l) 

300.000

200. 000 

100.000

20. 00 30. 00 

[min] 

Figure 9 GPC chromatogram of the sample solution (0 day, pH 9 n-1) 

15/20 

40. 00 
Study Number:8316490-00 

[mVl 

[mVl 

[mVl 

300.0

201000 

1 oo. o~8.l-,oo-o -------------::30:-:-:. o~o--------'---' 40. 00 

[min] 

Figure 10 GPC chromatogram of the sample solution (5 day, pH 4 n=ll 

300. 000 

200. 000 

100. 008 

2 . 00 30. 00 40. 00 

[min] 

Figure 11 GPC chromatogram of the sample solution (5 day. pH 7 n=ll 

300. 000 

200. 000 

100.000 

' 20. 00 30. 00 

[min] 

Figure 12 GPC chromatogram of the sample solution (5 day, pH 9 n=ll 

16/20 

40. 00 
Study Number:8316490-00 

7.2. Partition coefficient (n-octanollwater) 

~ 

0 

"-

.!l "' 

• 

1------~"l -1--------jY = 2.893x + 2.425 _ 

R2 = 0.963 

-0.700 -0.200 0.300 

logk 

Figure 13 Calibration curve 

17/20 

0.800 
Study Number:8316490-00 

[mVl 

[mVl 

150. OOOir---;::::=====-=-;----:---------:-----~ 

Thiourea 

' 100. 000 --------------------L------------------------L------------------------ 0 

' 

' 

' 

' 

' 

' 

' 

' ' 50. 000 --------------------r------------------------r------------------------

0. OOO•t=:"::;:::::==;===::;=:;:==;t===;===::;=:;:==;==;===::;=:;:==;===;====j 

0. 00 20. 00 40. 00 60. 00 

[min] 

Figure 14 HPLC chromatogram of the dead time solution 

300. 000-.----------~------~, 

~: 

-----------c 

T Toluene I : : 1-r=-=;J:.:_Ei.:• th.:..:yl:..::._be:..::nz.:..:en:..::._e~ ,------------ -!-----------------------

---------l :7-~----- -I Triphe~nylamine 1------------- - ---

' ' 

' ' 

200. 000 

100. 000 

' ' 

' ' 0. 000 F==:'=~="';=~;===r' =;===;====;=====r"--="='f===;====;=====;o==;c===j 

0. 00 20. 00 40. 00 60. 00 

[min] 

Figure 15 HPLC chromatogram of the standard solution 

18/20 
Study Number:8316490-00 

[mVl 

[mV] 

40. 000 

0. 00

QOO tOO 400 

40. 000 

lminl 

"·

0. 000 

000 tOO 400 

[min] 

Figure 16 HPLC chromatograms of the sample solution and blank solution (with no column) 

19/20 
Study Number:8316490-00 

[mV] 

[mVl 

40. 000 

20. 000 

0. 

0. 00 20. 00 40. 00 60. 00 

[min] 

Blank solution 

40. 000 ------,-----------------------·----------------------- ' 

20. 000- ---------------------- -t------------ -----------I-----------------------

' ' ' 

0.0001~~~-~~~~~~-~~~~~--~-

0. 00 20. 00 40. 00 60. 00 

[min[ 

Figure 17 HPLC chromatograms of the sample solution and blank solution (with a column) 

20/20